In the model of the A-factor (2-isocapryloyl-3R-hydroxymethyl-␥-butyrolactone) regulatory cascade in Streptomyces griseus, A-factor binds ArpA, the A-factor receptor protein, that has bound to the adpA promoter and dissociates it from the DNA, thus inducing the transcription of adpA. AdpA switches on the transcription of a number of genes required for secondary metabolism and morphological differentiation, forming an AdpA regulon. Consistent with this model, arpA null mutants produced streptomycin and a yellow pigment in larger amounts and formed aerial hyphae from an earlier growth stage than the wild-type strain. On the other hand, mutant MK2, expressing a mutant ArpA (Trp119Ala), neither produced secondary metabolites nor formed aerial hyphae, because this A-factor-insensitive mutant ArpA always bound to and repressed the adpA promoter due to the amino acid replacement of Trp-119 with Ala. Introduction of adpA under the control of a foreign promoter into mutant MK2 restored all of the phenotypes that we could observe, which suggests that the only significant target of ArpA is adpA. In contrast to other ␥-butyrolactone regulatory systems, disruption of arpA had no effect on A-factor production, indicating that ArpA does not regulate A-factor biosynthesis. Instead, A-factor production was found to be repressed by AdpA in a two-step regulatory feedback loop.
A Hanks-type protein kinase AfsK autophosphorylates on threonine residue(s) and phosphorylates AfsR, a global regulator for secondary metabolism in Streptomyces coelicolor A3(2). Mass spectrometry of a tryptic digest of the autophosphorylated form of AfsKD C corresponding to the kinase catalytic domain (Met-1 to Arg-311) of AfsK, together with subsequent site-directed mutagenesis of the candidate amino acids, identified threonine-168 as a single autophosphorylation site. Threonine-168 is located in the activation loop that is known for some Ser/Thr kinases to modulate kinase activity on phosphorylation of one or more threonine residues within the loop. Consistent with this, mutant T168D, in which Thr-168 was replaced by Asp, became a constitutively active kinase; it phosphorylated AfsR to the same extent as AfsKD C produced in and purified from Escherichia coli cells during which a considerable population of it had been already phosphorylated intermolecularly. All these findings show that autophosphorylation or intermolecular phosphorylation of threonine-168 in AfsK accounts for the self-activation of its kinase activity.Keywords serine/threonine kinase, autophosphorylation, self-activation, antibiotic production, Streptomyces A number of proteins in Streptomyces, including Streptomyces coelicolor A3(2), are phosphorylated on their serine/threonine and tyrosine residues in response to developmental phases [1,2]. Recent completion of the genome projects of S. coelicolor A3(2) [3] and S. avermitilis [4] has revealed the presence of about 40 proteins having a kinase catalytic domain similar to those of the typical eukaryotic serine/threonine and tyrosine kinases. All these observations clearly show that a given Streptomyces strain possesses several protein kinases of eukaryotic type, some of which regulate growth, morphological development and secondary metabolism. Of the protein serine/threonine kinases in Streptomyces, AfsK that phosphorylates serine/threonine residues of AfsR was first discovered, representing the first instance in the bacterial world in which the ability of a bacterial Hankstype protein kinase to phosphorylate an exogenous protein has been demonstrated [5]. The AfsK-AfsR system in S. coelicolor A3(2) globally controls the biosynthesis of secondary metabolites including actinorhodin, undecylprodigiosin, methylenomycin, and a calciumdependent antibiotic [2].Autophosphorylated amino acid residues of Hanks kinases in prokaryotes, such as PrkC in Bacillus subtilis [6], PknB in Mycobacterium tuberculosis [7ϳ9], PknD, PknE and PknF in M. tuberculosis [9], and PknH in M.
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